This revised application is in response to a Program Announcement for Exploratory and Developmental Research Grants for Investigations in Rare Diseases, which is funded through the R21 award mechanism. This project centers on the rare disease cystic fibrosis (CF), with focus on immunity to the bacterial pathogen Pseudomonas aeruginosa. In CF, infection with P. aeruginosa is linked to clinical deterioration. Antibiotics are steadily and alarmingly losing effectiveness due to resistance. The long-term goals of this project are to elucidate the defects in adaptive immunity toward P. aeruginosa that allow colonization to evolve into chronic lung disease in CF patients and to devise new therapies to correct these defects and vaccines to prevent infection. The major hypothesis being evaluated is that the failure to clear P. aeruginosa in CF is partly due to defective epithelial secretion of the chemokine RANTES, which results in ineffective opsonic antibody responses. We plan to investigate this hypothesis by analyzing the systemic and mucosal humoral immunity engendered by live-attenuated vaccine strains of P. aeruginosa delivered intranasally to transgenic CF mice. We have found that although this vaccination strategy can delay oropharyngeal P. aeruginosa colonization in CF mice, overall serum opsonic antibody levels are lower than those induced in wild type mice. In Aim 1, the mechanisms behind defective opsonic antibody production following nasal immunization of transgenic CF mice will be assessed with focus on RANTES expression using cytokine/chemokine protein arrays. Localization of labeled vaccine and expression of RANTES will also be evaluated using flow cytometry and confocal microscopic. In Aim 2, the immune effectors responsible for delayed oropharyngeal colonization of transgenic CF mice following nasal immunization will be characterized using passive immunotherapy. We will also investigate whether the protective efficacy of this immunization approach can be augmented by the use of RANTES as a mucosal adjuvant. Knowledge gained from these studies will elucidate new strategies for the prevention and treatment of P. aeruginosa infections in the setting of CF. [unreadable] [unreadable] [unreadable]